title: Tumour cells down-regulate CCN2 gene expression in co-cultured fibroblasts in a Smad7- and ERK-dependent manner.
abstract: BACKGROUND
  Recent studies have revealed that interactions between tumour cells and the surrounding stroma play an important role in facilitating tumour growth and invasion. Stromal fibroblasts produce most of the extracellular matrix components found in the stroma. The aim of this study was to investigate mechanisms involved in tumour cell-mediated regulation of extracellular matrix and adhesion molecules in co-cultured fibroblasts. To this end, microarray analysis was performed on CCD-1068SK human fibroblast cells after direct co-culture with MDA-MB-231 human breast tumour cells.
  
  
  RESULTS
  We found that the expression of both connective tissue growth factor (CTGF/CCN2) and type I collagen was negatively regulated in CCD-1068SK fibroblast cells under direct co-culture conditions. Further analysis revealed that Smad7, a known negative regulator of the Smad signalling pathway involved in CCN2 promoter regulation, was increased in directly co-cultured fibroblasts. Inhibition of Smad7 expression in CCD-1068SK fibroblasts resulted in increased CCN2 expression, while Smad7 overexpression had the opposite effect. Silencing CCN2 gene expression in fibroblasts led, in turn, to a decrease in type I collagen mRNA and protein levels. ERK signalling was also shown to be impaired in CCD-1068SK fibroblasts after direct co-culture with MDA-MB-231 tumour cells, with Smad7 overexpression in fibroblasts leading to a similar decrease in ERK activity. These effects were not, however, seen in fibroblasts that were indirectly co-cultured with tumour cells.
  
  
  CONCLUSIONS
  We therefore conclude that breast cancer cells require close contact with fibroblasts in order to upregulate Smad7 which, in turn, leads to decreased ERK signalling resulting in diminished expression of the stromal proteins CCN2 and type I collagen.
authors.affiliation: International Centre for Genetic Engineering and Biotechnology (ICGEB), Anzio Rd Observatory, Cape Town 7925, South Africa. iqbal.parker@icgeb.org.
authors.forename: Beverley A
authors.initials: BA
authors.lastname: van Rooyen
authors.forename: Georgia
authors.initials: G
authors.lastname: Schäfer
authors.forename: Virna D
authors.initials: VD
authors.lastname: Leaner
authors.forename: M Iqbal
authors.initials: MI
authors.lastname: Parker
mesh_terms: D001943:Breast Neoplasms / Q000378:metabolism*; D002460:Cell Line; D045744:Cell Line, Tumor; D018920:Coculture Techniques; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism*; D000090602:Collagen Type I, alpha 1 Chain; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005347:Fibroblasts; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D051906:Smad7 Protein / Q000235:genetics / Q000378:metabolism*
chemical_list: C526317:CCN2 protein, human; D024042:Collagen Type I; D000090602:Collagen Type I, alpha 1 Chain; C497806:SMAD7 protein, human; D051906:Smad7 Protein; D055513:Connective Tissue Growth Factor; D048049:Extracellular Signal-Regulated MAP Kinases; C522360:MMP1 protein, human; D020781:Matrix Metalloproteinase 1